Compared Anatomy of Young Leaves of Prunus persica (L.) Batsch with Different Degrees of Susceptibility to Taphrina deformans (Berk.) Tul

Anatomical observations of leaves infected by Taphrina deformans were studied in tolerant peach trees (TPT) and in very susceptible (VSPT) ones. Leaves from the first sampling (2nd April) showed hyphae penetrating through the stomata or into the cuticle of the host tissue; anatomical structures of leaf sections were similar for both TPT and VSPT. The ultrastructure of the leaves of TPT showed seemingly normal mesophyll cells. In contrast, mesophyll cells of the VSPT showed important signs of degradation. Cells were organelle‐free and the middle lamella was expanded and invaded by hyphae of T. deformans. In some samples, the leaves of TPT showed deformed epidermal cells, loss of some spongy cells and increase of the intercellular spaces and division of the palisade cells. The pathogen proliferation in the leaves of the VSPT was considerably superior. In this case, stimulation of cell division occurred in the abaxial epidermis. Cells showed periclinal and oblique divisions, with an increased number of plasmodesmata; palisade or spongy cells were not differentiable. Leaves from TPT collected on 26th April showed hyphae with a non‐cylindrical section and with a squashed aspect. The hyphae were very evident in the intercellular spaces, showing abundant endoplasmic reticulum of rough type (RER) in the cytoplasm. On the other hand, epidermis of the leaves of the VSPT had numerous hyphae under the cuticle, which were growing in a thick pectin matrix. Leaves from TPT and VSPT collected on 6th May showed relevant differences. The leaves of TPT had a palisade mesophyll with fewer cells but with active chloroplasts. In contrast, the leaves from VSPT showed empty mesophyll cells, the cytoplasm was collapsed and the adaxial epidermis was covered with the fungus fructification. The observed anatomical and ultrastructural differences of leaves from TPT and VSPT confirm a different behaviour in plant‐host reaction at early stages of infection.     

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

Degradation of pruning wastes by Phanerochaete sordida growing in SSF: Ultrastructural,chemical, and enzymatic studies

A solid standard fermentation (SSF) with the fungus Phanerochaete sordida in a medium with Nephrolepis cordifolia (entire pinnae separated from the rachis) and Laurus nobilis (fragmented leaves) was performed over 92 days to study the degradation of leaves with histological, chemical, and enzymatic methods. The fungus entered the leaves early, through the stomata in N. cordifolia and L. nobilis, and also through mechanical cuts that had been made in the latter. The initial attack affected the mesophyll in both plant species, and the phloem in L. nobilis. The vascular bundle of N. cordifolia was protected by a sheath of cells with thick lignified walls. The collenchyma cell walls situated near the principal vein in L. nobilis swelled during the initial stages of enzymatic action, but reduced their thickness afterwards, mainly in regions of contact with the hyphae. At the end of the experiment, no species had leaves with mesophyll. In L. nobilis, phloem was also lacking, and a partial and heterogeneous attack on the xylem became evident. The histological changes are compared with the enzymatic activities and the chemical composition of the culture media, describing the stages of fungal colonization.     

Leaf curl disease of Almond caused byTaphrina deformans (Berk.) Tul.

Summary The host/parasite relationship ofTaphrina deformans (Berk.) Tul. on Almond,Prunus dulcis (Miller) D. A. Webb (=Prunus amygdalus Stokes), has been studied with the light microscope by clearing and sectioning infected leaves.A quantitative study of the host reaction shows that the presence of the fungus causes immediate cell division (hyperplasia) followed by cell enlargement (hypertrophy) and cell differentiation. The epidermal and bundle sheath cells in infected regions contain anthocyanin.The vegetative mycelium is located in intercellular spaces in three distinct leaf regions. The sub-epidermal and intercellular hyphae are morphologically similar, consisting of an irregularly branching network of cells separated by unusual septa. Sub-cuticular hyphae have a more regular shape and become short and wide during development of the disease.Infection margins illustrate changes in the healthy leaf caused byT. deformans and observations indicate that the fungus spreads in the upper leaf regions.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

The ultrastructure of the development of Tillandsia (Bromeliaceae) trichome

Tillandsia spp. (Bromeliaceae) use their epidermal trichomes for absorbing atmospheric water, mineral and organic nutrients. The absorbing trichome in Tillandsia has a nail-like shape, formed by an axis (stem) connected to the internal tissues of the leaf, and by an external shield. Water and aqueous liquids coming from the external environment go through the shield cells and then run through the stem, finally reaching the underlying mesophyll parenchyma along a symplastic route.     

Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.     

Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants

The soilborne ascomycete fungus Verticillium dahliae causes destructive vascular wilt disease in hundreds of dicotyledonous plant species. However, our understanding of the early invasion from the epidermis to the vasculature and the prompt proliferation and colonization in the xylem tissues remains poor. To elaborate the detailed infection strategy of V. dahliae in host plants, we traced the whole infection process of V. dahliae by live-cell imaging combined with high-resolution scanning electron microscopy. The 4D image series demonstrated that the apex of invading hyphae becomes tapered and directly invades the intercellular space of root epidermal cells at the initial infection. Following successful epidermal invasion, the invading hyphae extend in the intercellular space of the root cortex toward the vascular tissues. Importantly, the high-resolution microscopic and live-cell images demonstrated (a) that conidia are formed via budding at the apex of the hyphae in the xylem vessels to promote systemic propagation vertically, and (b) that the hyphae freely cross adjacent xylem vessels through the intertracheary pits to achieve horizontal colonization. Our findings provide a solid cellular basis for future studies on both intracellular invasion and vascular colonization/proliferation during V. dahliae infection and pathogenesis in host plants.     

Biochemical characterization of cell types within leaves of metal-hyperaccumulating Noccaea praecox (Brassicaceae)

Background and aims

Distinct metal distribution patterns within leaves of metal hyperaccumulating plants are repeatedly observed however, the presumable role of key structural biochemical molecules in determining and regulating their allocation remains largely unknown. We aimed to characterise in a spatially resolved manner the distribution of the main biochemical components in leaves of field-collected Cd/Zn-hyperaccumulating Noccaea praecox in order to relate them to metal distribution patterns at tissue level.

Methods

The biomolecular composition of the leaves was spatially analysed using synchrotron radiation Fourier Transform Infrared (FTIR) and the distribution of Zn with synchrotron radiation Low-Energy X-Ray Fluorescence (LEXRF) microspectroscopy was determined on the same tissues of interest (epidermis, sub-epidermis, mesophyll).

Results

In epidermal cells high proportion of free-carboxyl, nitro and phosphate groups standing for pectin, nitroaromatics, phytic and other organic acids were found. Adjacent mesophyll cells had higher proportions of proteins, carbohydrates and cellulosic compounds.

Conclusions

Pectin compounds were indicated as important components of Zn enriched epidermal cell walls. In addition, intense lignification of epidermal cell walls might limit leakage of the trapped metals back to the metabolically active and thus more sensitive mesophyll. Distribution of metal-binding compounds in particular cell types/tissues may therefore predispose metal distribution patterns and tolerance in leaves.     

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state‐of‐the‐art quantitative 2D liquid chromatography/tandem mass spectrometry (2D‐LC‐MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size‐exclusion chromatography/inductively coupled plasma mass spectrometer (SEC‐ICP‐MS) method, in order to identify the Zn‐binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S‐transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn–nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn–nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type‐specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr

The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the β-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.     

Process of infection of armored scale insects (Diaspididae) by an entomopathogenic Cosmospora sp

Several species in the fungal genus Cosmospora (synonym Nectria) (anamorph Fusarium) are specialist entomopathogens of armored scale insects (Diaspididae), known to cause periodic epizootics in host populations. Inconsistent mortality rates recorded under laboratory conditions prompted a study into the process of infection of armored scale insects by this fungus. Scale insect mortality following exposure to a Cosmospora sp. (Culture Collection Number: CC89) from New Zealand was related to insect age, with reproductively mature insects having a significantly higher infection rate than immature insects. Examination using scanning electron microscopy found no evidence that the fungus penetrated directly through the wax test (cap) of the scale insect or through the un-lifted interface between the test and the substrate on which the insect resided. However, fungal hyphae were observed growing beneath the test when the test of the reproductively mature insect lifted away from the substrate for the purpose of releasing crawlers, the mobile pre-settled juveniles. Once the hyphae of CC89 advanced under the test, germ-tubes readily penetrated the insect body through a number of natural openings (e.g. spiracles, vulva, stylet), with mycosis observed within seven days after inoculation. Direct penetration through the cuticle of the scale insect was not observed.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.